Gram-negative bacteria frequently cause infections in humans. An abundant glycolipid found in the outer membrane of these bacteria, lipopolysaccharide (LPS), forms an integral part of their structure and modulates multiple interactions with the immune system during infection. It has become clear in recent years that many Gram-negative pathogens alter the structure of their LPS during adaptation to the host, in order to optimize immune evasion. This proposal seeks to apply novel experimental approaches to understand better the mechanisms and consequences of lipid A structural modification by Francisella during intracellular growth. This would represent the first detailed description of LPS adaptation in a cytosolic pathogen. Unlike the LPS of many medically-important Gram-negative microbes, Francisella LPS is non- inflammatory. It neither stimulates nor antagonizes LPS-sensing host machinery, and we have previously documented that it fails to interact with important LPS-sensing molecules that normally promote efficient elimination of Gram-negative bacteria. These unique characteristics likely derive from its unusual lipid A and core polysaccharide structures. The overall composition of Francisella LPS is also highly unusual in that a majority of the LPS molecules consist of free lipid A, lacking both core and O-antigen polysaccharides. The precise structural determinants of the inertness of Francisella LPS and the functional consequences of the presence of so much hydrophobic free lipid A are not fully understood. The presence of a large population of hydrophobic LPS molecules complicates some aspects of structural analysis, and in response to this challenge, we have developed a means to label replicating Francisella with [14C]acetate in both laboratory media and within human phagocytes. The label is incorporated into fatty acids, providing a sensitive, quantitative method to analyze the structure of Francisella LPS in detail. Mounting evidence indicates that among the adaptations made by Francisella as it invades and disseminates are changes in the structural composition of its lipid A/LPS. The methods we have developed to label, isolate, and quantify lipid A/LPS molecules based on specific structural characteristics will permit a detailed analysis of modulation of innate immunity by Francisella. Based on our preliminary data, we hypothesize that Francisella alters the structure of its lipid A during adaptation to the cytosol o mammalian cells, and that these changes result in a lipid A with distinct properties that facilitat evasion of innate immunity. To test this hypothesis, we propose the following specific aims: 1. Characterize the determinants of lipid A heterogeneity of F. tularensis 2. Determine how the formation of lipid A species accumulating selectively in infected host cells is regulated 3. Determine the functional consequences of the lipid A alterations that occur during intracellular infection